Fig 2: Sevoflurane exposure induces VIP cell hyperactivity in ACC of presymptomatic AD mice. (A) Schematic of ACC local circuitry. PYR, pyramidal neurons; PV, parvalbumin‐expressing interneurons; SST, somatostatin‐expressing interneurons; CRH, corticotropin‐releasing hormone neurons; VIP, vasoactive intestinal peptide‐expressing interneurons. (B) Experimental timeline and design for cell‐type‐specific expression of GCaMP6s in ACC of 5×FAD mice. (C) Example image of ACC showing VIP interneurons expressing GCaMP6s. (D) Representative image (left), Ca2+ traces (middle), and quantification of average Ca2+ activity in CRH neurons (n = 82, 117 cells from three mice per group, unpaired t test, t 197 = 0.3625, p = .7174). (E–G) Similar to (D), but for SST (E, n = 109, 132 cells from three mice per group, unpaired t test, t 239 = 0.2017, p = .8403), PV (F, n = 119, 132 cells from three mice per group, unpaired t test, t 249 = 1.93, p = .0547), and VIP interneurons (G, n = 193, 240 cells from five mice per group, unpaired t test, t 431 = 8.129, p < .0001). (H) Experimental timeline for chemogenetic inhibition of VIP cells in ACC of 5×FAD; Thy1‐GCaMP6s mice with recent exposure to sevoflurane. (I) Representative image of ACC region showing mCherry+ neurons. (J) Pyramidal neuron Ca2+ activity in ACC of 5×FAD mice expressing hM4Di in VIP cells (n = 280, 257 cells from four mice per group, unpaired t test, t 535 = 7.453, p < .0001). The dashed line indicates the Ca2+ level in 5×FAD mice with air exposure. (K) Inhibition of VIP cell hyperactivity improves recall performance in sevoflurane‐exposed 5×FAD mice (n = 6, 10 mice, unpaired t test, t 14 = 4.332, p = .0027). Summary data are presented as mean ± SEM. Each dot represents data from a single cell (D–G, J) or animal (K).

Fig 3: Cntnap1–/– mice showed increased spine density and an increase in the number of PV+ and VIP+ neurons.(A) Representative Golgi staining of cortical layers II/III of adult Cntnap1−/− mice (apical dendrites, 5 months of age, n = 3 for WT and Cntnap1−/− mice). Scale bar: 4 μm. (B) Costaining of Cntnap1 and PV in WT mouse brain at P15. Scale bar: 200 μm. (C) Representative immunochemical images of the S1 primary somatosensory cortex and hippocampus in coronal slices of P15 mouse brains stained with a PV antibody. Scale bar: 500 μm. (D) Quantitative analysis of spine density in Cntnap1–/– mice (22 and 25 neurons from each group were randomly selected and measured). Statistical significance was evaluated by Student’s t test. **P = 0.0093. Data are shown as mean ± SEM. (E and F) Quantitative analysis of the number of PV+ neurons in Cntnap1–/– mice (n = 3 mice for WT and KO) compared with their normal littermates. Statistical significance was evaluated by Student’s t test. Data are shown as mean ± SEM. For the quantification of PV+ neurons in cortex, ***P = 0.001. For the quantification of PV+ neurons in hippocampus, **P = 0.0025. (G and H) Representative immunochemical images of the S1 primary somatosensory cortex and hippocampus in coronal slices of P15 mouse brains stained with a VIP (green) and SST (red) antibody. Scale bar: 500 μm. On the right are enlarged images of boxed hippocampus stained with VIP antibody. (I and J) Quantitative analysis of the number of VIP+ and SST+ neurons in Cntnap1–/– mice compared with WT (n = 3 mice for WT and KO). For the quantification of VIP+ neurons in hippocampus, ***P = 0.0002. Statistical significance was evaluated by Student’s t test. Data are shown as mean ± SEM.

Fig 4: The circadian rhythm of Raptin release is controlled by SCNAVP neurons.a Schematic diagram of the retrograde tracing system in SCN-innervating PVNRCN2. AAV-Rcn2-Cre-EGFP, AAV-DIO-H2B-T2A-TVA and AAV-DIO-RVG were mixed and then bilaterally injected into PVN. Three weeks later, rabies virus (RV)-EnvA-tdTomato (red) were bilaterally injected into PVN. b, c Representative images (b) and quantification (c) of colocalization with RV-tdTomato (red) by immunofluorescence staining of AVP+ neurons (top line, green), VIP+ neurons (middle line, green) and GRP+ neurons (bottom line, green) in SCN. Scale bars, 50 μm (n = 3 per group). d Schematic diagram of the anterograde tracing system in SCN-innervating PVN. The WT mice were bilaterally injected with scAAV2/1-hSyn-Cre into SCN and AAV-DIO-mCherry into PVN. e, f Representative images (e) and quantification (f) of colocalization with mCherry (red) by fluorescence ISH of Rcn2 mRNA (green) in PVN neurons. Scale bar, 50 μm (n = 3). g Schematic diagram of chemogenetic inhibition of SCNAVP neuron of Avp-Cre mice. AAV-DIO or AAV-DIO-hM4Di was injected into SCN of Avp-Cre mice. Mice were intraperitoneally injected with CNO at a dose of 2 mg/kg body weight. h, i Representative images (h) and quantification (i) of co-staining of c-Fos (red) and RCN2 (green) in PVN after CNO injection for 60 min to induce SCNAVP neuron inhibition in Avp-Cre mice. Scale bar, 50 μm (n = 4 per group). j Plasma Raptin levels of mice injected with AAV-DIO or AAV-DIO-hM4Di before and after intraperitoneal injection of CNO for 1 h (n = 4 per group). k Schematic diagram illustrating the optogenetic activation of SCNAVP neuron terminal in PVN, followed by the detection of PVNRCN2 neuron activity. AAV-DIO or AAV-DIO.ChR2 was bilaterally injected into SCN, whereas AAV-Rcn2-Cre-EGFP was injected into the PVN of Avp-Cre mice. The fiber was implanted into the PVN to stimulate SCNAVP neuronal terminal. l, m Representative traces (l) and action potential frequency (m) of PVNRCN2 neurons in brain slice before and during optogenetic activation of SCNAVP neurons (n = 3 ~ 4 per group). n The plasma Raptin levels before and after optogenetic activation of SCNAVP neurons in vivo (n = 3 ~ 4 per group). o Schematic diagram illustrating the experimental workflow for SCNAVP neuron activation and PVNRCN2 neuron inhibition. AAV-DIO-mCherry or AAV-DIO-hM4Di-mCherry with AAV-Rcn2-Cre-EGFP were injected into PVN for chemogenetic manipulation of PVNRCN2 neuron. CNO was intraperitoneally injected into mice at a dose of 2 mg/kg body weight. AAV-fDIO.ChR2 with AAV-Avp-Flp were injected into SCN for optogenetic activation of SCNAVP neurons. The fiber was implanted into the PVN to stimulate SCNAVP neuronal terminal in PVN. p, q The plasma Raptin levels in mice injected with AAV-DIO (p) or AAV-DIO-hM4Di (q) before and after CNO treatment (n = 4 per group). Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01 by a two-tailed paired Student’s t-test (j, m, n, p, q) or by a two-tailed unpaired Student’s t-test (i). See also Supplementary information, Figs. S3, S4.

Fig 5: The effect of inhibition of ACC activity on nerve density and tumor progression in CRC mice.A Representative images of parasympathetic nerve markers VAChT staining in the colorectal tissue. VAChT (Green) and DAPI (blue). B Representative images of sympathetic nerve markers TH staining in the colorectal tissue. TH (Green) and DAPI (blue). Scale bar: 100 μm. C Quantitative of the density of VAChT-labeled parasympathetic nerve in the colorectal tissue across groups (n = 5,8,12,6 per group). D Quantitative of the density of TH-labeled sympathetic nerve in the colorectal tissue across groups (n = 5,10,12,6 per group). E Representative images of sensory nerve markers CGRP staining in the colorectal tissue. CGRP (Green) and DAPI (blue). F Representative images of peptiderergic nerve markers VIP staining in the colorectal tissue. VIP (Green) and DAPI (blue). Scale bar: 100 μm. G Quantitative of the density of CGRP-labeled sensory nerve in the colorectal tissue across groups (n = 4,10,8,6 per group). H Quantitative of the density of VIP-labelled peptiderergic nerve in the colorectal tissue across groups (n = 5,8,11,6 per group). I Representative images of cell proliferation markers Ki67 staining in the colorectal tumor tissue. Ki67 (Green) and DAPI (blue). Scale bar: 100 μm. J Representative images of apoptosis markers Cleaved-caspase3 staining in the colorectal tumor tissue. Cleaved-caspase3 (Green) and DAPI (blue). Scale bar: 100 μm. K Quantitative of the fluorescence intensity of the Ki67 in the colorectal tumor tissue across groups (n = 10,10,6 per group). L Quantitative of the fluorescence intensity of the Cleaved-caspase3 in the colorectal tumor tissue across groups (n = 11,10,5 per group). M Representative Western blot images showing the expression of Ki-67, PCNA, and cleaved caspase-3 in tumor tissues from CRC+Ctrl-ACC (Ctrl), CRC+Gi-ACC (Gi), and CRC+Gq-ACC (Gq) mice. N–P Quantification of Ki-67, PCNA, and cleaved caspase-3 protein levels normalized to β-Tublin in each group, (n = 5,5,6 per group; n = 5,5,6 per group, n = 5,6,6 per group). Q Representative images of tumor size in CRC+Ctrl-ACC, CRC+Gi-ACC and CRC+Gq-ACC groups. R Quantification of tumor volume in CRC+Ctrl-ACC, CRC+Gi-ACC and CRC+Gq-ACC groups, (n = 5,6,6 per group). All data are presented as mean ± s.e.m of the fold change of the Sham+Ctrl-ACC group and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test (C, D, G, H). All data are presented as mean ± s.e.m of the fold change of the CRC+Ctrl-ACC group and analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test (K, L, N–P, R). The source data are provided as Supplementary Data 1 in the Figshare repository.